Whilst this method can determine overall differences in permeability between the small and large intestine, it is unable to reveal detailed information relating to precise regional locations of altered integrity within each organ and also lacks standardisation. Additionally, external factors including gastrointestinal motility and mucosal blood flow may lead to inaccuracy of permeability measurements. The eponymously named Ussing chamber takes its name from Danish biologist Hans Ussing, who developed the technique in the 1950s to understand the phenomenon of active Na + transport. Such seminal studies paved the way for the present models of transepithelial transport, including the discovery of the Na +/K + ATPase pump.
In later years, the Ussing system helped unravel the mechanistic processes underpinning cystic fibrosis (CF). By monitoring ionic movements it was shown that Cl − secretion was decreased in patients, due to a mutated CFTR gene. The Ussing system offers an ex vivo measurement of permeability using fluorescent probes as well as electrophysiological measurements. The system design has been updated to accommodate multiple chambers as shown in Fig. The chambers support epithelial tissue or cell monolayers in such a way that each side of the membrane is isolated and faces a separate chamber-half. The potential difference (PD) across epithelial tissue can be determined using Ag/AgCl electrodes, giving an indicator of tissue health. Transepithelial resistance (TER) can be calculated to give an overall measurement of gut integrity. A low TER value is indicative of increased permeability. Short circuit current (Isc) is also often used during electrophysiological measurements. Isc refers to the current that is required to nullify tissue PD and is a summation of all ionic currents across the epithelium. Isc can be calculated from PD and TER values. Prior studies have shown that decreased TER under inflammatory conditions was associated with down regulation of “sealing” TJ proteins e.g. ZO-1, JAM-A and claudins-1, − 3 and − 5 as well as up-regulation of pore forming proteins (claudin-2 ). The use of fluorescently labelled probes such as 4 kDa FITC-dextran and lucifer yellow provide measurements of paracellular flux across the epithelium.
#Labchart reader finding slope differentiate software#
We describe in depth the modern multi-chambered Ussing system, with complementary software protocol.
Furthermore, we detail essential methods for dissecting and mounting tissue samples to ensure both viability and consistency of results.
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